2-nitrobenzyl proteins

Horton, H. R. Koshland, D. E. Jr. A highly reactive coloured reagent with selectivity for the tryptophan residue in proteins 2-hydroxy-5-nitrobenzyl bromide. J. Amer. Chem. Soc. 1965, 87, 1126-1132. 

Molecular imprinting is not limited to organic polymer matrices, but can also be applied to silica-based materials and even proteins. Proteins freeze-dried in the presence of a transition state analogue as template have been used successfully as catalysts, e.g., for the dehydrofluorination of a fluorobutanone. For instance, lyophilized 3-lactoglobulin imprinted in this manner with N-isopropyl-N-ni-trobenzyl-amine could accelerate the dehydrofluorination by a factor of 3.27 compared to the non-imprinted protein see Table 5 [62]. In a similar procedure, BSA was imprinted with N-methyl-N-(4-nitrobenzyl)-S-aminovaleric acid and showed an enhancement of the catalytic effect by a factor of 3.3 compared to the control protein for the same reaction see Table 5 [113]. 

The highly successful extension of site-directed suppression mutagenesis to a Xenopus oocyte system by the Dougherty and Lester labs discussed above has permitted structural, functional and kinetic studies of ion channels in intact cells [30-32, 62]. Recently, the introduction of residues that allow photochemical proteolysis and photo deprotection of protein side chains has been applied to the study of ion channels. Miller et al. introduced o-nitrobenzyl-pro-tected tyrosines at three positions in the a-subunit of nAChR [62 c]. The o-nitro-benzyl group was then removed photolytically to produce the wild-type residue during the course of a voltage-clamp study, so that time-resolved measurements could be made. 

H.R. Horton, H. Kelly and D.E. Koshland, Environmentally sensitive protein reagents 2-methoxy-5-nitrobenzyl bromide, J. Biol. Chem. 240 (1965) 722-724. 

Blawas AS, Oliver TF, Pirrung MC et al (1998) Step-and-repeat photopatteming of protein features using caged/biotin-BSA characterization and resolution. Langmuir 14 4243 250 Conrad DW, Golightley SK, Bart JC (1998) Photoactivatable o-nitrobenzyl polyethylene glycol-silane for the production of patterned biomolecular arrays. US Patent 5,773,308... 

Figure 5 Photolysis of 2-nitrobenzyl caged serine (a) and tyrosine (b) restores the wild-type residues. Photolysis of 2-nitrophenyl glycine (c) cleaves the protein backbone.

Slade and Vulfson have shown that the catalytic activity of native BSA in the dehydrofluorination reaction in aqueous media is greater than that reported for catalytic antibodies and molecularly imprinted polymers [30]. These authors therefore imprinted ]S-lactoglobulin and papain using A-isopropyl-4-nitrobenzyl amine as the transition state analogue. The catalytic activity of the imprinted proteins was evaluated in the dehydrofluorination reaction using acetonitrile as the reaction medium. A three fold rate enhancement in the A eat value vis-d-vis non-imprinted proteins was observed. 

Activity in nmol of product/min/100 mg of protein 27 C 15,000 X g supernatant fraction substrate, p-nitrobenzyl chloride. 

Proteins, characterization of tryptophane residues 2-Hydroxy-3-nitrobenzyl bromide. 

More recently, Yanagawa and coworkers has further improved the mRNA display method by incorporation of a photocleavable 2-nitrobenzyl linker between genotype (mRNA) and phenotype (protein) to facilitate isolation of the mRNA for amplification or analysis.295 They demonstrated the utility of the photocleavable linker by also isolating FKBPla from an mRNA library using biotinylated FK506. 

Other Methods of Ionization. There are several other methods for ionization in addition to ESI and MALDI. However, most of them are not commonly used in proteomics. Some of these include chemical ionization, electron ionization, fast atom bombardment (FAB), and many others. Most of these lead to disintegration or fragmentation of analyte molecules and are not commonly used in proteomics. However, FAB has some application in the analysis of proteins and peptides, because this is a soft ionization procedure and does not cause the fragmentation of molecules under analysis. In the FAB method, a nonvolatile matrix such as m-nitrobenzyle alcohol is used to hold the analyte molecules. Analyte molecules are vaporized and ionized by bombardment with the high-energy beam of xenon or cesium from a probe inserted directly into the device containing the sample. Ionized molecules thus obtained are then subjected to separation by the mass... 

Preparation of caged proteins by introduction of an o-nitrobenzyl group directed toward specific residues dates back to the mid-1990s. In a pilot study, bovine serum albumin (BSA) was randomly labeled with up to 15... 

The intermediacy of a complex has been proposed to account for the high specificity of 2-hydroxy-5-nitrobenzyl bromide (XVIII) for tryptophan in a reaction with proteins . There is no spectroscopic evidence for this complex at present. 

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